!1 ELECTRO-PHARMACO-PATHOPHYSIOLOGY OF CARDIAC ION CHANNELS AND IMPACT ON THE ELECTROCARDIOGRAM By ANDRÉS RICARDO PEREZ RIERA MD Key words: Ion channels – channelopathies – electrocardiogram RESTING POTENTIAL, ELECTROLYTIC CONCENTRATION, FAST AND SLOW FIBERS, ACTION POTENTIAL PHASES, SARCOLEMMAL AND INTRACELLULAR CHANNELS Introduction If we place both electrodes or wires (A and B) from a galvanometer (a device that records the difference in electric potential between two points) in the exterior (extracellular milieu) of a cardiac cell in rest or polarized, we would see that the needle of the device does not move (it indicates zero), because both electrodes are sensing the same milieu (extracellular). That is to say, there is no difference in potential between both ends of the galvanometer electrodes: Figure 1. !2 FIGURE 1 ILLUSTRATION OF TRANSMEMBRANE RESTING OR DIASTOLIC POTENTIAL IN A CARDIAC CELL AND MEASUREMENT WITH GALVANOMETER ! In rest, the extracellular milieu is predominantly positive in comparison to the intracellular one, as a consequence of positive charge (cations) predominance in the first in comparison to the intracellular one. What is the reason for the extracellular milieu to be predominantly positive in comparison to the intracellular? Reply: The reason lies in the greater concentration of proteins existing in the intracellular milieu in comparison to the extracellular one. Proteins have a double charge (positive or negative), and for this reason they are called amphoteric (amphoteric is any substance that can behave either as an acid or as a base, depending on the reactive agent present. If in the presence of an acid, it behaves as a base; if in the presence of a base, it behaves as an acid); therefore, in the intracellular pH negative charges are predominantly dissociated; i.e. proteins behave as anions (-) in the intracellular milieu. Table 1 shows the normal concentrations of cations and anions in the extra and intracellular milieus. In this table, it is seen that the predominant cation within the cell is K+ and in the extracellular milieu, Na+. !3 TABLE 1 NORMAL ELECTROLYTIC CONCENTRATION IN THE EXTRA AND INTRACELLULAR MILIEUS ELECTROLYTE EXTRACELULLA INTRACELULLA Extracelullar/ R R intracellular E/I ratio Na+ 135 to 145 mEq/L 10 mEq/L 14:1 K+ 3.5 to 5.5 mEq/L 155 mEq/l 1: 30 Ca2+ 2 mEq/L 10-4 2 x 10-4 = 1. Although intracellular Ca2+ concentration is 2 mM, most is attached or isolated in the mitochondria and the SR. Mg2+ 2 mEq/L 15 mEq/L 0.1333 Cl- 95 to 110 mEq/L 20 to 30 mEq/L 4:1 CO3H+ 27 mEq/L 8 mEq/L 3.3 Proteins 2 mEq/L 90 mEq/l 0.022 If we place the ends of both electrodes (A and B) in the intracellular milieu, the galvanometer needle will remain in zero (Figure 2), because both electrodes are in the same milieu. Figure 2. !4 FIGURE 2 ILLUSTRATION OF TRANSMEMBRANE RESTING POTENTIAL IN CARDIAC CELLS AND MEASUREMENT WITH GALVANOMETER ! Finally, if one of the ends is placed within the cell (wire B) and the other is in the extracellular milieu (wire A), we observe that the needle moves, indicating the difference in potentials between the extracellular (+) and intracellular (-) milieu, with a value of ≈ – 90 mV, indicating the existence of a difference in potentials of -90 mV. Figure 3. FIGURE 3 ILLUSTRATION OF TRANSMEMBRANE DIASTOLIC OR RESTING POTENTIAL IN CARDIAC CELLS AND MEASUREMENT WITH GALVANOMETER !5 ! This value of -90 mV, represents the difference in Diastolic Transmembrante Potential (DTP) or resting potential; i.e. the difference in potential existing during diastole between the extracellular milieu with predominantly positive charges (+) and the intracellular milieu with predominantly negative charges (-). Considering the profile image of the action potential (AP) of cardiac cells or cardiac fibers are grouped into rapid and slow: I) Rapid cells: Present in automatic Purkinje cells and nonautomatic cells of the ventricular and atrial contractile myocardium (“ordinary working muscle cells”). In Figure 4, the characteristics of the AP profile of rapid fibers, and their main responsible ionic channels are outlined. FIGURE 4 PROFILE OF RAPID FIBER AP AND MAIN ION CHANNELS !6 ! • Rapid inward Na+ (in phase 0). • Early inward K+ by the Ito channel in phase 1. • Slow inward Ca2+ in phase 2. • End delayed inward K+ in phase 3, made up by two or three slow delayed outward K+ channels: slow (Iks), rapid (Ikr) and ultra-rapid (Ikur). The latter originates in the hKv1.5 channel (1). Kv1.5 channels conduct the delayed ultra-rapid current of the rectifyier K+ channel, Ikur. In human beings, Kv1.5 channels are highly expressed (are present) in the atria, but are scant (or absent) in the ventricles (2). • Finally, the Na+/K+ ATPase pump acts in phase 4. This pump, with the energy consumption, places three Na+ cations in the extracellular milieu, and introduces a K+ ion. The pump is inhibited by digitalis (3). • Pacemaker current, If or “funny” current: it operates exclusively in the initial portion of phase 4 (it only acts in a potential range from -60/-70 mV to -40 mV) and contributes 20% to determine the heart rate (HR) of P cells of the SA node (4), controlling diastolic depolarization and spontaneous activity of P, pacemaker cells. The molecular determinants of the If channel, belong to a family of channels activated in hyperpolarization known as HCN channels, made up by 4 isoforms (HCN1, HCN2, HCN3, HCN4), with HCN2 (chromosome 19p13.3) and !7 HCN4 being the main ones in the heart (Hyperpolarization-activated Cyclic Nucleotide-gated channels family (HCN)). Based on the sequence of HCN channels, these are classified as members of a superfamily of voltage-gated K+ (Kv) and CNG channels (5;6). A research showed that inhibiting the If current could be used to decrease the incidence of coronary artery disease (CAD) in a subset of patients with HR≥70 bpm (7;8). The mutations in HCN4 (chromosome 15q24-125.3) and CNBD (S672R) isoforms are associated to familial inherited bradycardia, as they cause an effect similar to parasympathetic stimulus, by reducing If channel activity (9). There are micro-domains of the membrane, rich in cholesterol and sphingolipids in cardiomyocytes, called caveolae. In caveolin-3 (CAV3), several channels have been located, such as L-type Ca2+, INa+ (Na(v)1.5), the If pacemaker channel (HCN4) or the Na+/Ca2+ exchanger (NCX1) and others. Mutations in CAV3 may originate variant 9 of congenital Long QT Syndrome (LQT9) and other inherited arrhythmias. In acquired diseases that lead to congestive heart failure, CAV3 may be affected, originating arrhythmias (10). The fast Ca2+ T type current or T-type Ca2+ channel, transient ICa-T current or tiny conductance channel, voltage-dependent T-type calcium channel, and low-voltage-activated (T-type) calcium channels are responsible for the entrance of Ca2+ in the final part of phase 4 in the SA Node, in the N region of the AV node and in the His-Purkinje System. The rapid type Ca2+ channel is blocked in a selective way by the Ca2+ antagonist mibefradil (mibefradil-sensitive component), and other drugs such as bepridil, flunarizine, and pimozide, which bind to the receptor channel in a concentration-dependent fashion, thus blocking the Ca2+ cation entrance. Mibefradil decreases HR, not affecting contractility (11). The great efficacy of bepridil to end with atrial fibrillation or flutter, is due in part to the block of this rapid type Ca2+ channel (12). ICa-T is not sensitive to dihydropyridine. ICa-T has its function increased with noradrenaline, the α adrenergic agonist phenylephrine, the ratio of extracellular ATP and endothelin-1 (ET-1). !8 II) Slow cells: Located within the limits of the SA Node of Keith and Flack, atrioventricular node, and in the mitro-tricuspid rings. They are characterized by presenting a not so negative resting potential (≈ -55 mV), a not so wide, slow Ca2+- dependent phase 0, and with additional final entrance of Na+ through a channel independent from voltage, called INa+B, and absence of identifiable phases 2 and 3. Figure 5 shows the typical outline of a slow fiber, with the main operating channels. !9 FIGURE 5 PROFILE OF AP OF SLOW FIBER AND MAIN ION CHANNELS ! The main differences between rapid and slow fibers are summarized in Table 2. Rapid fibers are Purkinje cells and muscle cells from the atria and ventricles, and slow fibers are constituted by P cells from the SA Node, nodal cells, atrioventricular or AV Node cells, and mitro-tricuspid rings. TABLE 2 MAIN DIFFERENCES BETWEEN RAPID AND SLOW FIBERS RAPID FIBERS SLOW FIBERS Location: Atrial and ventricular muscle, inter-nodal SA node, AV preferential pathways (of Bachmann, node, and Wenckebach, and Thorel), His-Purkinje mitro- System (HPS). tricuspid rings. Kinetics: Rapid: activation and inactivation <1 ms. <5 ms and inactivation between 3 -80 ms. !10 Diastolic resting -90 to -80 Mv. -60 mV potential: Activation or -70 to -55 mV. -55 to -30 potential mV. threshold AP amplitude in 100 to 130 mV. At 35 to 70 mV: mV. Activation in <1 ms. <5 ms. ms: Inactivation in <1 ms 3-80 ms. ms: Phase 0 Tetrodotoxin (TTX) Ca2+ blockers: Anti-arrhythmic agents class I: IA, IB and antagonists, IC. some divalent cations such as cadmium (Cd), manganese (Mn), cobalt (Co) and nickel (Ni). Type of All-or-nothing type. Dependent response to on the stimulus: intensity of the stimulus applied. Dromotropism: 0.3 to 3 or 5 M/s (Meters per second) 0.01 to 0.10 M/s. Overshoot: +20 mV. It could be absent or up to +15 mV. SA Node: Absent. Present. !11 Atrial Present.